1. Field of the Invention
This invention relates to methods of detecting genetic and phenotypic markers in biological samples using spectral imaging and brightfield microscopy to detect the presence of chromogenic dyes.
2. Background Art
In cytopathological diagnostic laboratories, cytological specimens are routinely stained with permanent dyes such as hematoxylin and eosin and for decades, pathologists have based their diagnosis of disease on cyto- and histological features as seen under a light microscope. Unlike fluorescent dyes, permanent dyes do not fade or bleach so that second opinion diagnosis, re-examination of archived material and even retrospective studies, can be performed. Thus, for routine cytopathological diagnostic purposes, fluorescence microscopy is not preferred for these reasons as well as because of high auto-fluorescence inherent to the tissue type or which might be induced by fixation.
Immunohistochemical and in situ hybridization methods have become increasingly important for research and diagnosis of disease. Also, multi-parameter cytochemical analysis is required when rare or unique material is to be studied. Many fluorescent markers with emission spectra ranging from blue to infra-red have become available for multi-color detection due to advances made in conjugation chemistry. Thus, although fluorescence microscopy could be used for these multi-parameter applications, for the above-mentioned reasons, it is often not possible to use methods employing fluorescence.
The present invention overcomes previous shortcomings in the art by providing methods for analyzing both genetic and phenotypic markers in a single biological sample through the use of bright field spectral imaging of chromogenic dyes. Such analyses are valuable in a variety of clinical applications, such as, for example, the diagnosis and characterization of cancer and the analysis of chromosomal aberrations in pre- and post-natal diagnostics.
An important aspect of the present invention that overcomes a severe limitation in the art is that by using the methods provided herein, multiple probes, both to genetic and/or phenotypic markers, and therefore multiple chromogenic dyes can be used in the same sample and the individual dyes can be distinguished using spectral imaging, even where the sample has been previously stained with a cytological stain which otherwise would obscure the signal from the genetic or phenotypic probes. Using these methods, a pathologist for example, can stain a tissue sample to observe a general morphological aspect of cells in the sample, and a geneticist can subsequently use that stained sample to diagnose cells in the sample for the presence of a genetic or phenotypic marker, such as a chromosomal aberration associated with cervical cancer, with much more clarity, accuracy, ease, and efficiency than using previously available methods.